Benzyloxycarbonyl compounds are known in the literature as protecting groups for amines and in some examples as a protecting group for hydroxyl moieties. This group of compounds has been used in solid phase peptide synthesis (SPPS) because of the ease in which the group is removed through hydrogenation or hydrobromic acid treatment. Similarly, thiocarbonates are known and have been reviewed. Various chemical protection and deprotection strategies have been provided by Berger et al. (J. Am. Chem. Soc. 1956, 78, 4483), Zervas et al. (J. Am. Chem. Soc. 1963, 85, 1337), Sokolovsky et al. (J. Am. Chem. Soc. 1964, 78, 1202).
A previous study explored the use of an S-isobutyloxymethyl protective group. Young et al. (Young, G. T.; Brownlee, P. J. E.; Cox, M. E.; Handford, B. O.; Marsden, J. C. J. Chem. Soc. 1964, 3832) discovered that this group was stable to 2 N hydrochloric acid and 50% acetic acid, and decomposed slightly in 2 N sodium hydroxide. Hiskey et al. (Hiskey, R. G.; Sparrow, J. T. J. Org. Chem. 1970, 35, 215-220) teaches that cysteines in SPPS may be protected during chain elongation with the S-isobutyloxymethyl protective group. This group was stable in 12 N hydrochloric acid in acetone, warm aqueous acetic acid and hydrazine hydrate in refluxing ethanol. It showed decomposition to silver nitrate in ethanol and boron trifluoride etherate in acetic acid.
None of these references was applied to oligonucleotide synthesis nor did the references address the sensitivities of the deprotection strategies. In fact, the current invention demonstrated that the S-benzyloxycarbonyl (S-CBz) thiol-modified six-carbon linker was stable to concentrated ammonia (30% v/v) for ˜18 h, but was completely hydrolyzed by dilute sodium hydroxide in short periods. This was an unexpected result.
Dellinger et al (U.S. Ser. No. 11/903,821; US 2009/0082554) teaches the use of thiocarbonate linkers for polynucleotides. These inventors employ the thiocarbonate moiety as cleavable linkers between an oligonucleotide and protein, ligand (consists of a drug carrier, drug, targeting molecule, or molecules that can improve intercellular or intracellular transport) or a solid support used for oligonucleotide synthesis such as TENTA gel. It is not clear from their teachings that this thiocarbonate linkage could be used to protect a sulfur atom when applying a thiol modifier at the 5′-terminus of the oligonucleotide and upon nucleophilic release post-oligonucleotide synthesis enables the 5′-thiol-modified oligonucleotide to participate in a Michael reaction with a Michael acceptor molecule.
Nokihara et al. (Nokihara, K.; Berndt, H. J. Org. Chem. 1978, 43, 4893-4895) teaches that the carbobenzoxy (CBz) group may be used to protect disulfide forms (sulfenyl) of cysteine in peptide synthesis. It is stable to trifluoroacetic acid treatment and mild alkali conditions. It is unstable to strong alkali treatment. It is not clear from their teachings that this group could be used for oligonucleotide synthesis and its deprotection scheme.
Currently, several reagents are available for the addition of thiol groups on an oligonucleotide. One is S-Bz TEG-CE phosphoramidite (Link Technologies, Item Number: 2187, United Kingdom). This phosphoramidite synthon is protected by the benzoyl (Bz) group.
This current invention comprises the S-benzyloxycarbonyl (S-CBz) group instead of the thiobenzoyl (S-Bz) group. Studies have shown that that the S-Bz group is too labile to hydrolysis and would not remain attached to the oligonucleotide during deprotection of the phosphate groups and bases. Because there is no selective deprotection, the fully deprotected oligonucleotide with thiol could undergo dimerization (disulfide formation) at the appropriate pH.
The 5′-thiol modifier, C6 (S-trityl-6-mercaptohexyl-(2-cyanoethyl)-N,N′-diisopropyl-phosphoramidite (Glen Research, item number 10-1926-xx) uses the standard phosphoramidite chemistry for insertion and is stable to premature removal due to the robustness of the triphenylmethyl (trityl)-derivatized thioether bond. To deprotect the thiol group, a heavy metal is required. Silver nitrate solution (1 M) is used at room temperature for 30 minutes.
This current invention does not require a heavy metal for deprotection. As a result, there would be no need to remove the metal from the reaction solution, thereby facilitating the final purification.
The 5′-thiol modifier, C6 S—S (1-O-dimethoxytrityl-hexyl-disulfide, 1′-[(2-cyanoethyl)-(N,N′-diisopropyl)]-phosphoramidite (Glen Research, item number 10-1936-xx) uses the standard phosphoramidite chemistry for insertion and is stable to premature removal due to the robustness of the disulfide bond (as indicated by the S—S term in the name). To deprotect the thiol group, a reducing agent is required, such a β-mercaptoethanol, dithiothreitol (DTT) or tris(carboxyethyl)phosphine (TCEP). Typically, a concentrated DTT solution (˜0.5 M) is used at room temperature or slightly elevated temperature for 30 minutes. The by-product of this reduction is a mole equivalent of the protecting thiol counterpart which must be removed or sequestered prior to use for subsequent conjugation reactions (e.g. Michael reaction).
This current invention does not yield such reactive by-products in the deprotection reaction or process. As a result, there would be facilitation of the final purification.
Reactive by-products are also produced in β-elimination reactions where the sulfur is protected by a thioether. As a result of basic or acidic treatment, the by-product could be an alkene. If not carefully removed, this unsaturated product may react with the thiol. An example of this would be the following study.
West et al. (West, C. W.; Estiarte, M. A.; Rich, D. H. Org. Lett. 2001, 3(8), 1205-1208) teaches that the 9-fluorenylmethyloxycarbonyl (FMOC) group may be used in the protection of the cysteine side chain in solid phase peptide synthesis (SPPS. They link an aromatic moiety to the sulfur through a thiocarbonate group. As the investigators point out, this strategy has a drawback that re-deployment of the aromatic fluorine moiety can ensue to form an alkylated species linked through a thioether bond. The introduction of a fluorine group into the synthesis route with oligonucleotides can cause immediate termination of the synthesis as this group is removed under conditions too harsh to maintain the integrity of the oligomer.
In addition, De La Torre et al. (de la Torre, B. G.; Avino, A. M.; Escarceller, M.; Royo, M.; Albericio, F.; Eritja, R. Nucleosides & Nucleotides 1993, 12(9), 993-1005) teach that the base-labile group, dinitrophenylethyl (DNPE) can be successfully employed for the purpose of oligonucleotide synthesis. A series of oligonucleotides were synthesized and modified with a linker containing this functionality. The by-product of this reaction is dinitrophenylethylene, which may re-react with the thiol if not removed or sequestered.
This present invention was designed to avoid this recombination, as the by-product of the deprotection reaction is a benzyl alcohol. Moreover, due to the lack of β hydrogens with the benzyl group, there is no possibility of reactive unsaturated compounds forming.
Common methodologies to install a sulfur-containing group into an oligonucleotide sequence entails the use of either a triphenylmethyl (trityl group, linked through an acid-labile thioether or a 4,4′-dimethoxytriphenyl C6 alkyl group, linked through disulfide. Functionally, the steric bulk of the trityl group protects the sulfur, while the lipophilicity of the three phenyl moieties aids in the purification by reverse phase methods. The disulfide synthon has wide scale applicability for use as a 3′-modifier, 5′-modifier or internal modifier. The installation of these agents is accomplished through the standard phosphoramidite chemistry.
However, to remove the trityl group, deprotection methods require the presence of heavy metals. Moreover under these conditions, there is a greater risk of oligonucleotide depurination. To ease the conditions necessary for deprotection, monomethoxytrityl (MMT) and dimethoxytrityl (DMT) groups have been explored. While the DMT on oxygen is well established as the optimized 5′-hydroxyl moiety, its utility as a thiol-protecting group is limited by the nature of the sulfur atom resulting in greater sensitivity to DNA synthesis conditions.
The disulfide group is homolytically cleaved by reducing agents such as dithiothreitol (DTT) or tris(carboxy)ethyl phosphine (TCEP) to liberate the thiol. The by-product of homolytic cleavage would be another thiol, which, if not removed by chromatography or other means, could react with target molecule and could reduce the overall yield by 50%. Therefore, a new protecting group was sought, as these strategies were considered inappropriate.
Despite the utility of these protecting groups, the literature is deficient in base labile protecting groups for sulfur. Clearly, these protecting groups are not applicable or suitable for all cases. Specifically, this invention describes the use of the CBz group and outlines the use of modified CBz groups for the protection of a thiol functionality in oligonucleotide synthesis. Additional aromatic and alkyl thiocarbonates are included. Examples are provided to support the use of the CBz-protecting group as a 5′-modifier in oligonucleotides.